High-density, high throughput biological and biochemical assays have become essential tools for diagnostic and research applications, particularly in areas involving the acquisition and analysis of genetic information. These assays typically involve the use of solid substrates. Examples of typical quantitative assays performed on solid substrates include measurement of an antigen by ELISA or the determination of mRNA levels by hybridization. Solid substrates can take any form though typically they fall into two categories—those using spherical beads or those using planar arrays.
Planar objects such as slide- or chip-based arrays offer the advantage of allowing capture molecules, e.g., antibody or cDNA, of known identity to be bound at spatially distinct positions. Surfaces are easily washed to remove unbound material. A single mixture of analytes can be captured on a surface and detected using a common marker, e.g., fluorescent dye. The identification of captured analytes is governed by the spatial position of the bound capture molecule. Archival storage of the array is generally possible. Because the array corresponds to a stationary flat surface, detection devices are generally simpler in design and have lower cost of manufacture than bead reading devices. One of the difficulties of the planar array approach is the initial positioning of the capture molecule onto the surface. Techniques such as robotic deposition (e.g., “Quantitative monitoring of gene expression patterns with a complementary DNA microarray” by Schena et al. Science, 270:467-470 (1995)), photolithography (e.g., “Light-directed, spatially addressable parallel chemical synthesis” by Fodor et. al. Science, 251:767-773 (1991)), or ink-jet technologies (e.g., “High-density oligonucleotide arrays” by Blanchard et al. Biosensors Bioelectronics, 6/7:687-690 (1996)) are generally used. These methods have a number of limitations. They require expensive instrumentation to generate high density arrays (greater than 1000 features/cm2), and there is no ability to alter the pattern after manufacture, e.g., replace one capture cDNA with another, consequently any alterations require a new manufacturing process and greatly increase expenses. Moreover, molecules bound to large flat surfaces exhibit less favorable reaction kinetics than do molecules that are free in solution.
One way around many of these problems is to use surfaces of small particles. Spherical beads have been the small particles of choice because of their uniform symmetry and their minimal self-interacting surface. Small particles, however, suffer from the problem of being difficult to distinguish, e.g., a mixture of beads is not spatially distinct. A number of technologies have been developed to overcome this problem by encoding beads to make them distinguishable. Companies such as the Luminex Corporation have developed methods of doing this by incorporating different mixtures of fluorescent dyes into beads to make them optically distinguishable. In a similar manner, other researchers have developed ways of incorporating other optically distinguishable materials into beads (e.g., “Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules” by Han et al. Nature Biotechnology, 19:631-635 (2001)). Furthermore, quantum dots, nanometer scale particles that are neither small molecules nor bulk solids, have also been used for bead identification. Their composition and small size (a few hundred to a few thousand atoms) give these dots extraordinary optical properties that can be readily customized by changing the size or composition of the dots. Quantum dots absorb light, then quickly re-emit the light but in a different color. The most important property is that the color of quantum dots—both in absorption and emission—can be “tuned” to any chosen wavelength by simply changing their size. Genicon Sciences Corporation (Their “RLS” particles are of nano-sizes and have certain “resonance light scattering (RLS) properties) also developed micro-beads or nano-beads with optically distinguishable properties. However, in using any of these approaches, it is difficult to manufacture more than 1,000 or so different encoded beads.
Beads are also the format of choice in combinatorial chemistry. Using the one-bead/one-compound procedure (also known as the split and mix procedure) (see “The “one-bead-one-compound” combinatorial library method” by Lam et al. Chem. Rev., 97:411-448 (1997)), it is possible to generate huge libraries containing in excess of 108 different molecules. However, the beads are not distinguishable in any way other than by identifying the compound on a particular bead. Labeled “tea bags” which contain groups of beads displaying the same compound have been used to distinguish beads. Recently, IRORI has extended the tea bag technology to small canisters containing either a radiofrequency transponder or an optically encoded surface. This technology is generally limited to constructing libraries on the order of 10,000 compounds, a single canister occupies ˜0.25 mL. Moreover, the technology is not well suited to high-throughput-screening. PharmaSeq, Inc. uses individual substrates containing transponders. These devices are 250μ×250μ×100μ. Larger libraries can be synthesized directly onto a surface to form planar arrays using photolithographic methods (such as those used by Affymetrix). However, such techniques have largely been restricted to short oligonucleotides due to cost considerations and the lower repetitive yields associated with photochemical synthesis procedures (see e.g., “The efficiency of light-directed synthesis of DNA arrays on glass substrates” by Mc Gall et al. J. Am. Chem. Soc., 119:5081-5090 (1997)). In addition, the available number of photo-labile protecting groups is severely limited compared to the tremendous breadth and diversity of chemically labile protecting groups that have been developed over the past 30+ years for use on beads. Recently, SmartBeads Technologies has introduced microfabricated particles (e.g., strip particles having dimensions of 100μ×10μ×1μ) containing bar codes that can be decoded using a flow-based reader. Microfabricated particles have the advantage that a nearly infinite number of encoding patterns can be easily incorporated into them. The difficulty lies in being able to easily analyze mixtures of encoded particles. Since such particles tend to be flat objects as opposed to spherical beads, they tend to be more prone to aggregation or overlapping as well as being more difficult to disperse.
Nicewarner-Pena et al., Science, 294(5540):137-41 (2001) recently reported synthesis of multimetal microrods intrinsically encoded with submicrometer stripes. According to Nicewarner-Pena et al., complex striping patterns are readily prepared by sequential electrochemical deposition of metal ions into templates with uniformly sized pores. The differential reflectivity of adjacent stripes enables identification of the striping patterns by conventional light microscopy. This readout mechanism does not interfere with the use of fluorescence for detection of analytes bound to particles by affinity capture, as demonstrated by DNA and protein bioassays.
A system incorporating the advantages of planar arrays and of encoded micro-particles would address many of the problems inherent in the existing approaches. Illumina, Inc. has attempted to do this by providing a method of generating arrays of microbeads using etched glass fibers (e.g., “High-density fiber-optic DNA random microsphere array” by Ferguson et al. Anal. Chem., 72:5618-5624 (2000)). However, Illumina's oligonucleotide based fluorescent-encoding microbeads are also limited in the number of unique representations. BioArray Solutions has used Light-controlled Electrokinetic Assembly of Particles near Surfaces (LEAPS) to form arrays of beads on surfaces (WO 97/40385). However, the LEAPS approach is still subject to the same restrictions as bead-based techniques with respect to the types of available encoding.
There exists needs in the art for microdevices and methods that can take the advantages of both microfabricated particles and spatially distinct arrays. This invention address these and other related needs in the art.